Hello HOPEians,
Please find below the action items from the RF3 meeting on June 5, 2023. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar.
Actions: (Please let us know if we can cross any of these off the list)
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Fran and Ulrike meet to discuss JLats updates
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Kumar Lab – Send the Ott lab the mutated sequences (Sequencing clones in Jurkats, VLPs to express base editors that can mutate and fight the response) Ursula Schulze-Gahmen can test for TAR structures
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Kumar lab – Test if you can use dead Cas9 for base editing
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Ott and Kumar lab – Send Ulrike the constructs to test. Schedule a call to test this
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Niren – Combine and put a PEG coding on the PLGA, use conjugation coding and do this at the beginning. Try more extensive pegylation
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Kumar lab- Connect with Ursula for biochemistry for host gene targeting
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Bring Esper onto RF3 to address their global solutions concerns
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Michael – Share mice tissue flash freezing protocols with Priti
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Priti can test materials sent
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Niren – Send LNPs to Priti for the Kumar lab to test – Transfection of primary human t cells with aptamer target LNPs with heart tissues
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Lish – work on MTA for this (Brec, latency models), Ulrike to send MTA contact officer to Lish
Updates:
Rubens Almeida-Tavora: Homing Endonucleases
Rubens shared his work on the use of homing endonucleases to inactive adeno-associated virus transgene expression. They are using the Adeno-associated virus (rAAV) as an in vivo gene therapy platform. rAAV has broad tropism, low immunogenicity, and lack of pathogenesis but can also achieve the long-term expression of transgenes. AAV gene therapy can be used to treat hemophilia A.
Rubens shared a study on the functionally cured “Miami Monkey”. Rhesus macaques infected with SHIV and AAV delivery of monoclonal antibodies showed little to no SHIV viral load. There are pros and cons to the long-term expression of AAV. It is currently impossible to halt AAV expression in case of adverse events. To inactivate AAV transgene expression, we have turned to Homing Endonucleases.
Homing Endonucleases (HEs) are naturally expressed “selfish” mobile genetic elements found across a wide range of organisms. They promote their own propagation through targeted recombination and have many characteristics that are perfect for our goal. In order to screen naturally occurring HE’s, Rubens and colleagues utilized a luciferase-based platform. They compared the toxicity of their three best HEs and designed inactive endonucleases to serve as negative controls. To help improve their target construct, they will change the location and add more targets.
Rubens shared the paper published in Nature on “Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes”. The study showed that placing introns within the coding region of a gene can significantly increase transgene expression. Using this technology, Rubens and colleagues were able to make improvements to the target construction by implementing new intron technology for Anil-Y2, Bmol, and Ppol reporters.
Additionally, they introduced an Anil-Y2 within the OSM signal sequence to further improve the target reporter. This can significantly contribute to transgene inactivation. Rubens explained further the design of mRNA expressing HE, LNP delivered HEs toxicity profile, alternative approaches, and the design of their in vivo experiments.
Other Updates:
We also heard quick updates from Ulrike, Fran, and Yaping. Ulrike shared data from their focus meetings on production for specific AAVs in mice. They are looking for biodistribution and want to boost expression for Brec1. Fran shared the Lenti usable dock system for brec1. The doc-inducible dBrec1 and Brecoff express and bind to HIV LTR. They will introduce Doxo and find out why it is not working in JLats. Yaping is working with primary t-cells and improving efficiency on the CCR5 and TAR base editing. The single base mutation was confirmed from sequencing data and it will be hard to transfect cells CCR14.
In the next RF3 meeting, we will hear more updates from Fran on jLat results and from Yaping on CCR5 and TAR base editing data.
Announcements
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Check out the Community Jam Board to help address some of our community’s concerns
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The HOPE Collaboratory-wide meeting is July 24th 9am PT/12pm ET..
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The next RF3 meetings is August 7th
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Upcoming Speakers:
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June 15 Dr. Mimi Ghosh
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June 26 Drs. Steven Deeks & Michael Peluso
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Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.
“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”
“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”